Project Summary/Abstract The rationale for this project is based on the need for improved strategies for functional and aesthetic reconstruction of craniofacial defects caused by trauma, tumor removal, infection, congenital malformations, or other diseases. Due to many tissue types in close proximity and a nearby bacteria-filled oral cavity, repair of craniofacial tissue is complex. Therefore, the objective of this study is to develop novel approaches and to improve upon existing strategies for craniofacial bone tissue repair by utilizing a scaffold to promote soft tissue healing and space maintenance of the defect, local antibiotic release to combat infection, and tissue growth of specified geometry at a distant site within the body to eliminate donor-site morbidity associated with autograft for repair. The proposed research will test the fundamental hypothesis that local release of antibiotics will eliminate local infection, restore soft tissue healing at the defect site, and allow robust bone growth at a distal site. The proposed research will be accomplished through two specific aims: 1) To manufacture and characterize properties of space maintainers utilizing different antibiotic-loading methods to understand kinetics of antibiotic release and to ensure mechanical properties are adequate for mandibular implantation and 2) To evaluate the effects of an antibiotic-releasing space maintainer on mandibular infection in an in vivo large animal model while bone is grown adjacent to rib periosteum in a 3D printed bioreactor. The space maintainers will be evaluated via mechanical testing (compression, 4-point bending, screw pull-out) and microcomputed tomography (microCT) (for pore size and porosity). The released antibiotic concentration will be evaluated via high performance liquid chromatography (HPLC). Bacteria will be utilized to determine minimum inhibitory concentration of antibiotic. In order to evaluate the effects of the space maintainer in vivo, the mandibular site will be swabbed (for identification, testing of potential antibiotic resistance) and will be analyzed by microCT for bone growth and histology for tissue and cell types. The tissue grown in bioreactors will be analyzed by microCT and by histology for bone growth, with quantitative polymerase chain reaction (qPCR), and via compression and screw pull-out testing. Blood drawn at several time points will be utilized to detect systemic markers of infection, oral swabs will be cultured, and vitals (such as heart rate and temperature) will be monitored. Upon completion of these studies, the expected outcomes are the successful fabrication of a space maintainer capable of antibiotic release with robust mechanical properties, the successful utilization of 3D printed bioreactors in a large animal model, and the successful development of a large animal model with sustained, yet localized, mandibular infection. In addition to improving upon existing strategies, this work will broaden our understanding on the interplay between bacteria, immune response, antibiotic delivery, and bone growth. Moreover, the proposed system provides a therapeutic approach for complex defects of large or unusual sizes that cannot utilize traditional autograft tissues due to shape or size constraints.